(RXR)-Selective Agonists: Analogues of 4 - ACS Publications

Sep 3, 2016 - X Receptor (RXR)-Selective Agonists: Analogues of 4‑[1-(3,5,5,8,8-. Pentamethyl-5,6,7 ... retinoid X receptors (hRXRs), with one or mo...
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Modeling, Synthesis, and Biological Evaluation of Potential Retinoid X Receptor (RXR)-Selective Agonists: Analogues of 4‑[1-(3,5,5,8,8Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and 6‑(Ethyl(5,5,8,8-tetrahydronaphthalen-2yl)amino)nicotinic Acid (NEt-TMN) Michael C. Heck,†,∥ Carl E. Wagner,†,∥ Pritika H. Shahani,† Mairi MacNeill,† Aleksandra Grozic,† Tamana Darwaiz,† Micah Shimabuku,† David G. Deans,† Nathan M. Robinson,† Samer H. Salama,† Joseph W. Ziller,‡ Ning Ma,§ Arjan van der Vaart,§ Pamela A. Marshall,† and Peter W. Jurutka*,† †

School of Mathematical and Natural Sciences, New College of Interdisciplinary Arts and Sciences, Arizona State University, 4701 West Thunderbird Road, Glendale, Arizona 85306, United States ‡ Department of Chemistry, University of California, Irvine, 576 Rowland Hall, Irvine, California 92697, United States § Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, CHE 205, Tampa, Florida 33620, United States S Supporting Information *

ABSTRACT: Sulfonic acid analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene, 1) as well as seven novel and two reported analogues of 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN) were synthesized and assessed for selective retinoid X receptor (RXR) agonism. Compound 1 is FDA-approved for treatment of cutaneous T-cell lymphoma (CTCL); however, 1 can provoke side effects by impacting RXR-dependent receptor pathways. All of the analogues in this study were evaluated for their potential to bind RXR through modeling and then assayed in an RXR−RXR mammalian-2-hybrid (M2H) system and in RXR-responsive element (RXRE)-mediated transcriptional experiments. The EC50 profiles for these unique analogues and their analogous effectiveness to inhibit proliferation in CTCL cells relative to 1 suggest that these compounds possess similar or even enhanced therapeutic potential. Several compounds also displayed more selective RXR activation with minimal cross-signaling of the retinoic acid receptor. These results suggest that modifications of potent RXR agonists such as NEt-TMN can lead to improved biological selectivity and potency compared with the known therapeutic.



INTRODUCTION

X receptor (LXR), to name only a few. All nuclear receptors modulate gene expression, generally promoting transcription, when signaled by a specific receptor ligand. This ligand, often a natural endogenous molecule, associates with the receptor by binding to a receptor ligand-binding domain (LBD), precipitating a conformational change that permits high-affinity receptor binding to the receptor’s corresponding hormone-

There are three known isoforms (α, β, γ) of the human retinoid X receptors (hRXRs), with one or more being expressed in every human tissue type, that primarily function as versatile transcription factors. The RXRs often act as heterodimeric partners with other receptors of the nuclear receptor (NR) superfamily comprising transcriptional regulators that include the retinoic acid receptor (RAR), the thyroid hormone receptor (TR), the peroxisome proliferator-activated receptor (PPAR), the vitamin D receptor (VDR), and the liver 1

© 2016 American Chemical Society

Received: May 30, 2016 Published: September 3, 2016 8924

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than simple RXR sequestration. Nevertheless, two key issues in developing therapeutic rexinoids with decreased pleiotropic effects concern RXR potency and heterodimer selectivity. Hence, an approach to modify the structure of the central RXR heterodimer agonist may lead to specific NR modulators (SNuRMs) that exhibit unique characteristics arising from varying NR control.15 Rexinoid SNuRMs are attractive medicinal drug targets because activating RXR selectively versus RAR bestows chemotherapeutic effects in an array of human cancer types without incurring toxicities arising from RAR crossover.16 Following lengthy development and testing17 of 9-cis-RA modeled compounds, 4-[1-(3,5,5,8,8-pentamethyltetralin-2-yl)ethynyl]benzoic acid (1)18 (Figure 1) was demonstrated to

responsive element (HRE) on the DNA. While HREs have increasingly been located either up- or downstream a considerable distance from the genes they regulate, many HREs have been found close to or within the regulated gene promoter region. The HREs exhibit sequence specificity, typically consisting of two hexad repeat sequences made up of half-sites punctuated by a variable number of nucleotide spacers between these inverted, direct, or everted repeats.2 The corresponding HREs for VDR, TR, and RAR contain half-sites separated with three, four, and five nucleotide spacers, respectively.3 While VDR, TR, and RAR were first believed to bind as homodimers to their corresponding HREs,4 they actually partner with RXR to form heterodimers in order to bind to their HREs.5 When one of several natural RXR agonist ligands such as 9-cis-retinoic acid (9-cis-RA) binds to RXR, an RXR homodimer self-assembles that then binds to RXR-responsive elements (RXREs). When RXR partners with other NRs in heterodimers, it does so either with bound or unbound ligand within the RXR LBD. With respect to the RXR−VDR heterodimer, RXR has been reported to be unoccupied.6 Alternatively, evidence suggests that RXR possesses a ligand to function as an RXR−LXR heterodimer.7 In view of this versatility, it is not surprising that RXR has been classified as the primary NR regulator, since it is often a prerequisite for heterodimer formation and proper transcriptional activation of other NRs’ corresponding response elements.8 A multitude of studies employing selective RXR ligands (rexinoids), RXR, and a variety of different nuclear receptors have resulted in two primary classifications for RXR− heterodimer complexes, namely, permissive and nonpermissive heterodimers. Only ligands selective for the RXR partner can activate purely nonpermissive RXR heterodimers, whereas either RXR-specific or partner-selective ligands can activate permissive RXR heterodimers.9 The generally nonpermissive RXR heterodimers include RXR−VDR, RXR−RAR, and RXR− TR. In most (but not in all) circumstances, RXR is “silent” in VDR and TR heterodimers. The RXR−RAR heterodimer, however, shows increased activity with select RXR agonists in combination with an RAR ligand. In fact, a few rexinoids have been reported to stimulate RXR−RAR despite the absence of a RAR-specific ligand.10 In this respect, RXR−RAR has been termed conditionally nonpermissive. Finally, the RXR−PPAR, RXR−LXR, and RXR−FXR heterodimers have been observed to be fully permissive. Developing RXR-specific agonists for clinical therapeutic treatments is complicated not only by potential pleiotropic effects arising from the activation of permissive RXR heterodimers but also from a limited RXR pool that may be titrated away from RXR-containing heterodimers to instead form RXR homodimers in certain cell conditions and tissue types, thereby limiting the ability of the nonpermissive RXR heterodimers to form and function. For instance, RXR agonists such as 9-cis-RA can arrest nonpermissive heterodimer activation in VDR11 or TR.12 Moreover, NR agonists such as T3 or 1,25-dihydroxyvitamin D3 (1,25D), which promote RXR heterodimer formation with TR and VDR, respectively, may also drive crosstalk inhibition of other RXR-dependent receptors utilizing the limited RXR pool. In fact, this crosstalk inhibition has been observed for T3−TR−RXR-directed inhibition of VDR transactivation13 as well as 1,25D−VDR− RXR-mediated inhibition of TR function;14,12 however, the cross-receptor squelching mechanism likely extends further

Figure 1. Two synthetic rexinoids, 1 and 2, modeled after 9-cis-retinoic acid.

possesses high RXR agonist specificity, and Ligand Pharmaceuticals Inc. eventually gained FDA approval for the drug, more commonly called bexarotene, for cutaneous T-cell lymphoma (CTCL) treatment. Many structurally similar bexarotene analogues, such as disilabexarotene (2),19 have been reported over the last 20 years, and many of these rexinoids possess RXR-specific agonism similar to that of bexarotene. While bexarotene (1) was approved as a treatment for CTCL, it has been explored for therapeutic effects in colon cancer20 and mammary carcinoma,21 and a proof-of-concept (POC) clinical trial has shown it to be effective in nonsmall cell lung cancer22 since upregulation of certain RXR-controlled genes suppresses cell proliferation while synergizing apoptosis in cancer cells subjected to chemotherapeutics.20 Several rexinoids, following compound 1, were investigated in noninsulin-dependent diabetes mellitus (NIDDM) mouse models because the RXR−PPAR heterodimer impacts metabolism.23 Despite bexarotene’s RXR specificity versus RAR, its known side effects include hypothyroidism,24 hyperlipidemia, and cutaneous toxicity. Like 9-cis-RA, side effects of bexarotene arise from disrupting nonpermissive receptor activation, as in TR,25 resulting in hypothyroidism, or stimulating permissive receptor activation, as in LXR, leading to hyperlipidemia26 or in RAR effecting cutaneous toxicity27 with therapeutic dose concentrations. Hence, the approach to design, model, and synthesize more potent and selective rexinoids whose pleiotropic effects are attenuated is an active area of research. Adding to the motivation to create novel rexinoids, RXRselective agonists have recently been investigated in neurodegenerative diseases such as Parkinson’s disease,28 and a recent POC trial of bexarotene in patients with mild to moderate Alzheimer’s disease (AD)driven by controversial murine studies in AD29revealed provocative results in critical human biomarkers in the clinic.30 Many RXR-specific ligands modeled after 9-cis-RA and 1 have been reported (Figure 2). For instance, cyclopropyldienoic acid 331 has been shown to be more potent as an RXR agonist than its enantiomer. Compound 9cUAB30 (4)32 is in 8925

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effects of hypothyroidism and triglyceridemia are often managed with hormone replacement therapy and statins. Because of the selective and potent RXR agonism observed for compounds 1 and 20−22, our laboratories have recently focused on preparing the seven novel analogous compounds 23−29, modeled after compounds 20−22, as well as the sulfonic acid analogues of bexarotene, 30 and 31 (Figure 3).

Figure 3. New rexinoids 23−31 prepared and evaluated in the present study.

These novel rexinoids were subsequently assessed for RXR activation and EC50 profile, RAR crossover, potential for causing hypertriglyceridemia, ability to inhibit cancer cell proliferation, and mutagenicity in side-by-side assays with 1 and compounds 20−22.

Figure 2. Additional synthetic rexinoids modeled after 9-cis-retinoic acid.



advanced clinical trials for breast cancer,33 and many 9cUAB30 analogues34 have also been described. Aryltrienoic acid RXR agonists, either unbranched,35 locked by one ring,36 or with multiple-fused37 rings, were reported by Boehm and coworkers, and 5 exemplifies the last category. The substitution of a fluorine atom for a hydrogen atom adjacent to the acid group on 1 gives compound 6,38 and substituting fluorine for the last remaining hydrogen atom adjacent to the acid group on 6 gives compound 7.39 These two compounds show increased RXR activation in the HCT-116 and Caco-2 cell lines, respectively. Substituting a heteroaromatic pyridine ring for the benzene ring of 1 gives compound 8,40 and modifying the bridging alkene group of 8 to a bridging cyclopropyl group gives compound 9 (LGD100268),40 which shows greater potency than 1 in CV-1 cells.40 Compound 1041 possesses an unsaturation in the aliphatic ring of 1 and has been produced in kilogram quantities.42 Acrylic acids 1143 (CD3254) and 1244 (CD2915) are also potent RXR agonists. Our group recently reported rexinoids 13−1945 as novel analogues of 8−11, and several of these analogues exhibited differential side effect profiles and gene expression in vivo.46 Recently, Kakuta and coworkers described the potent diarylamine carboxylic acid 6(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (20),47 more commonly called NEtTMN,47 and several assays characterized this compound as a potent RXR agonist. Compounds analogous to 20 that have been reported include diarylamine 2148 and its methylated analogue 22.48a,b Kakuta and co-workers used 20 as a starting point to develop partial agonists to serve as diabetes therapeutics without the side effect profile that comes from treatment with a potent RXR agonist. However, in diseases such as cancer, there is still a great need to develop additional potent RXR agonists. While weighing the side effect profiles and therapeutic potential for compounds, we must be cognizant that more effective therapeutics are often approved despite the potential for side effects. In the case of potent RXR agonists, the more severe side

RESULTS AND DISCUSSION Molecular Modeling. Docking scores for compounds 20− 32 relative to bexarotene are shown in Figure 4; each circle

Figure 4. Docking results for compounds 20−31 relative to bexarotene. For each compound and for bexarotene, docking to 15 different RXR ligand-binding domain structures was performed. Each circle represents the difference in binding free energy between the best pose of the compound and the best pose of bexarotene in one of the RXR structures; negative values indicate better binding of the compound than of bexarotene. (A) Autodock charges. (B) OpenBabel charges.

represents the best pose to a particular crystal structure, and ΔG < 0 indicates more favorable predicted binding of the compound than of bexarotene. The averaged calculated docking scores were within 1.2 kcal/mol of that for bexarotene, with the most favorable binding for compounds 30 and 31. The poses of the docked compounds were largely similar to each other (this is illustrated for compounds 20 and 23 in Figure 5), 8926

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acidification with 20% hydrochloric acid to give 4-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoic acid (22) in 71% yield (Scheme 1). Again, a transparent crystal of 44 was given from chloroform, and an Xray diffraction study revealed disorder in the aliphatic ring system (see the Supporting Information). The synthesis of the novel compound 6-(ethyl(3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (23) could not be accomplished starting with an approach of coupling amine 42 to 34 through a nucleophilic aromatic substitution reaction with conditions similar to those used in the synthesis of 20running this type of reaction, we simply recovered 42 and 34. Hence, we used the palladium coupling approach, whereby 6-bromo-1,1,4,4,7-pentamethyl-1,2,3,4-tetrahydronaphthalene (45)45 was coupled to methyl 6-aminonicotinate (46) to give methyl 6-((3,5,5,8,8-pentamethyl5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinate (47) in 37% purified yield. Methyl ester 47 was ethylated under standard substitution conditions to give methyl 6-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinate (48) in 78% purified yield. Methyl ester 48 was saponified in methanolic potassium hydroxide followed by acidification to give 6-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8tetrahydronaphthalen-2-yl)amino)nicotinic acid (23) in 91% yield (Scheme 2). A route similar to that of Kakuta and co-workers to make NEt-TMN47a was followed to make the novel compound 2(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylic acid (24). Amine 33 was mixed with methyl 2-chloropyrimidine-5-carboxylate (49) and pTsOH and refluxed in 1,4-dioxane for 18 h to give methyl 2((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylate (50) in 75% yield. Amine 50 in DMF was ethylated to give methyl 2-(ethyl(5,5,8,8tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylate (51) in 75% yield. Finally, methyl ester 51 was saponified to obtain 24 in 97% yield (Scheme 3). The novel pyrimidine compound 2-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5carboxylic acid (25), on the other hand, was prepared using the palladium coupling methodology. Aryl bromide 45 was coupled to methyl 2-aminopyrimidine-5-carboxylate (52) in the usual palladium(0) catalytic system to give methyl 2-((3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylate (53) in 45% purified yield. Methyl ester 53 was ethylated to give methyl 2-(ethyl(3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylate (54) in 65% purified yield. Methyl

Figure 5. Top poses for 20 (blue) and 23 (violet) docked to the RXR binding pocket.

with the fused-ring systems in nearly identical positions and small shifts in the positions of the single rings and bridgeheads to optimize binding. The acidic groups on the single ring of the compounds were observed to hydrogen-bond to Arg 315. Chemistry. The route of Kakuta and co-workers was followed to reproduce NEt-TMN (20)47a from commercially available 1,1,4,4-tetramethyl-6-nitro-1,2,3,4-tetrahydronaphthalene (32), and details about synthetic intermediates en route to 20 (33−36) are provided in the Supporting Information. The known compound 4-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoic acid (21) was prepared using a palladium coupling method reported by Kakuta and co-workers,47c and experimental procedures for the synthetic intermediates en route to 21 (37−39) are also provided in the Supporting Information. Fortuitously, compounds 20 and 21 both formed transparent single crystals from DMSO. Hence, Xray diffraction studies were performed and revealed the structures of 20incorporating DMSO within the crystal in a 1:1 ratioand 21 (see the Supporting Information). The known compound 4-(ethyl(3,5,5,8,8-pentamethyl5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoic acid (22) was synthesized in a similar method as 21. The method of Kakuta and co-workers was used to nitrate 40 with nitric acid in acetic anhydride to give 4147e in 50.4% yield, and 41 was subsequently reduced by palladium-catalyzed reduction in a flow reactor49 to give amine 4247e in 97% yield. Next, amine 42 was coupled to 37 in a palladium(0) catalytic system to give methyl 4-((3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen2-yl)amino)benzoate (43) in 81% purified yield. Methyl ester 43 was treated with sodium hydride in DMF followed by ethyl iodide to give methyl 4-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8tetrahydronaphthalen-2-yl)amino)benzoate (44) in 97% purified yield. The methyl ester of 44 was saponified under standard methanolic alkaline conditions at reflux followed by

Scheme 1. Shared Synthetic Route for Compounds 22, 28, and 29

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Scheme 2. Shared Synthetic Route for Compounds 23, 25, and 27

Scheme 3. Shared Synthetic Route for Compounds 24 and 26

ester 54 was saponified to give 25 in 84% yield after acidification and purification (Scheme 2). The pyrazine analogue of NEt-TMN, 5-(ethyl(5,5,8,8tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine2-carboxylic acid (26), was synthesized in an analogous manner by means of the same methodology as used by Kakuta.47a Amine 33 was reacted with methyl 5-chloropyrazine-2carboxylate (55) and p-TsOH by refluxing in 1,4-dioxane for 18 h to give methyl 5-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylate (56) in 56% yield. Amine 56 in DMF was deprotonated with sodium hydride, and the resulting anion was reacted with ethyl iodide to produce methyl 5-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylate (57) in 51% yield. Finally, methyl ester 57 was saponified under standard conditions to obtain 26 in 71% yield (Scheme 3). Again, we alternately employed palladium coupling to access the novel compound 5-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylic acid (27). First, aryl bromide 45 was coupled to methyl 5aminopyrazine-2-carboxylate (58) to give methyl 5((3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylate (59) in 63% purified yield. Methyl ester 59 was treated with sodium hydride in DMF followed by ethyl iodide to give methyl 5-(ethyl(3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylate (60) in 25% purified yield. Methyl ester 60 was saponified under the usual conditions to provide 27 in 57% yield (Scheme 2). Again, we were fortunate to isolate transparent crystals of 26 and 60, and X-ray diffraction studies were performed (see the Supporting Information). To probe how fluorination of 21 affects its activity, we prepared 4-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)-2-fluorobenzoic acid (28) in a palladium coupling reaction starting from commercially available 2-fluoro-

4-iodobenzoic acid (61). Benzoic acid 61 was converted to its methyl ester 62 by slow addition of thionyl chloride to a solution of 61 in methanol at 0 °C followed by reflux for 1 h in a modified version of the method of Kakuta and co-workers.47c Subsequently, amine 33 was coupled to 62 in the standard palladium(0) catalytic system to give methyl 2-fluoro-4((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoate (63) in 52% purified yield. Methyl ester 63 was ethylated to provide methyl 4-(ethyl(5,5,8,8-tetramethyl5,6,7,8-tetrahydronaphthalen-2-yl)amino)-2-fluorobenzoate (64) in 68% purified yield, and 64 was saponified to make 28 in 85% yield after isolation (Scheme 1). We were pleased to recover a single crystal of 28 from DMSO, and the X-ray diffraction study structure is provided in the Supporting Information. To complete our preparation of NEt-TMN analogues, the novel compound 4-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)-2-fluorobenzoic acid (29) was synthesized according to the route shown in Scheme 1. Amine 42 was coupled to 62 by palladium to access methyl 2-fluoro-4((3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoate (65) in 65% purified yield. Methyl ester 65 was ethylated with ethyl iodide to afford methyl 4-(ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)-2-fluorobenzoate (66) in 91% purified yield. Methyl ester 66 was saponified to give 29 in 84% yield (Scheme 1). Because of the excellent predicted binding of 4-(1-(3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzenesulfonic acid (30), the sulfonic acid analogue of 1, we also set out to synthesize 30 and its ketone analogue 4(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene-2carbonyl)benzenesulfonic acid (31). Commercially available potassium 4-carboxybenzenesulfonate (67) was refluxed in thionyl chloride and thereby converted to 4-(chlorosulfonyl)benzoyl chloride (68). Benzoyl chloride 68 was mixed with 40, 8928

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Scheme 4. Synthetic Route to Sulfonic Acid Bexarotene Analogues 30 and 31

Figure 6. Identification of RXR agonists via a mammalian two-hybrid screening assay in human colon cancer cells. HCT-116 human colon cancer cells were cotransfected using pCMV-hRXR binding domain vector (BD), pCMV-hRXR activation domain (AD), pFR-Luc reporter gene containing BD-binding sites, and renilla control plasmid. Cells were transfected for 24 h utilizing a liposome-mediated transfection protocol and then treated with either ethanol or DMSO vehicle or analogues at (A) 25 nM, (B) 100 nM, or (C) 1000 nM for 24 h. Analogue-mediated RXR binding and homodimerization, as measured by luciferase output, was compared with that of RXR agonist parent compound 1, which was set to 100%.

was produced in 48.9% yield. Vinylsulfonate 71 was refluxed with KOH in methanol and then treated with 20% HCl to give 30 in 80% yield (Scheme 4). Biological Assays and Rationale. Mammalian TwoHybrid Assay Demonstrates That Several Novel Analogues Induce RXR Homodimerization. Biological evaluation of the synthetic analogues described above (compounds 20−31) was first conducted using the mammalian two-hybrid system in the human colorectal carcinoma cell line HCT116. In this assay, ligand binding to a recombinant RXR containing an activation domain induces dimerization with an RXR−Gal4 fusion protein. The subsequent dimer then binds to multiple Gal4

and when aluminum chloride was added to the mixture, Friedel−Crafts acylation provided 4-(3,5,5,8,8-pentamethyl5,6,7,8-tetrahydronaphthalene-2-carbonyl)benzene-1-sulfonyl chloride (69) in 86% yield. Sulfonyl chloride 69 was converted to 31 in 56% yield by treatment of 69 with sodium hydroxide in acetone followed by acidification with 20% HCl. Sulfonyl chloride 69 was also observed to react with 2-methyl-2propanol in acetone and triethylamine to give isobutyl 4(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene-2carbonyl)benzenesulfonate (70) in 87% yield. When isobutyl sulfonate ester 70 was treated with a solution of triphenylphosphine methylide, isobutyl 4-(1-(3,5,5,8,8-pentamethyl5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzenesulfonate (71) 8929

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illustrated with compounds 31 and 30. Also, fluorination does not alter homodimerization, as shown by testing of compounds 29 and 28 compared to 22 and 21. Determination of EC50 Values. We utilized the mammalian two-hybrid system in HCT116 colon cancer cells to determine the EC50 values for compounds 20−31. Full dose−response curves were generated using a wide range of analogue concentrations (0.1−2000 nM for compounds 20−29 and 1− 80 μM for compounds 31 and 30). The results revealed that compounds 20, 23, 24, 25, 29, and 22 have EC50 values lower than that of compound 1 (13.8, 13.8, 40.9, 18.2, 33.8, and 7.9 nM, respectively; Table 1). These results are not surprising because these compounds exhibited greater RXR homodimerization at 25 nM compared with compound 1 (Figure 6A). In contrast, compounds 26, 27, 28, and 21 have EC50 values that were 2.4−7-fold higher than that of compound 1. As observed in the mammalian two-hybrid assay (Figure 6), acidification of the parent compound 1 to generate 30 and 31 resulted in decreased homodimerization, and the EC50 for compound 30 was determined to be approximately 18 200 nM which is 350fold higher than that of compound 1, while an EC50 value for 31 could not be calculated even when dosing at 80 000 nM. The EC50s for 30 and 31 were surprising, since docking predicted highly favorable binding (Figure 4) with hydrogen bonding between the sulfonate of the compounds and Arg 315 of the RXR (residue numbering as in Protein Data Bank (PDB) entry 1MVC50). This disagreement could stem from the implicit solvent, which might mistreat dehydration.51 Alternatively, compounds 30 and 31 may bind to the RXR but not activate it, a profile that would be more indicative of an antagonist. Therefore, we conducted competitive binding experiments in which we treated HCT-116 cells with increasing concentrations of 30 and 31 and a standard concentration of bexarotene, and these experiments revealed decreasing bexarotene-directed activation of RXR in the presence of increasing concentrations 30 and 31 (Figure S21 in the Supporting Information). These results suggest that 30 and 31 likely reach the binding pocket of RXR, but they appear to exert antagonism compared with 1. Finally, it is interesting to note that compounds 20 and 23 exhibit almost identical EC50 values (Table 1), and the best docked poses (Figure 5) suggest very similar modes of binding despite the structural difference of a methyl substitution. Novel Analogues Are Able To Direct RXR/RXRE Homodimer-Mediated Transcription. The mammalian two-hybrid assay is a useful initial screen for RXR-agonist-induced homodimerization because of its accessibility, speed, and sensitivity. However, it is necessary to determine whether RXR agonists are able to induce transcription in a natural protein and DNA configuration. Therefore, we performed a second screening assay that utilizes transfection of human RXRα and an authentic RXRE from the rat cellular retinol binding protein II gene. As with the mammalian two-hybrid assay, three concentrations of compounds were used (25, 100, and 1000 nM). At 25 nM (Figure 7A), compounds 20, 23, 24, 25, 29, 22, and 28 exhibited increased RXR-mediated transcriptional activity compared with compound 1, with the values being amplified by 27−66%. The greatest activity was observed with compounds 20, 23, 24, and 25. These results are similar to those obtained in the mammalian two-hybrid system (Figure 6A). Taken together, the results suggest that at 25 nM compounds 20, 23, 24, 25, 29, 22, and 28 are more efficient than compound 1 at binding to RXR and inducing RXR

DNA response elements, which are used to drive the expression of firefly luciferase. We tested the indicated compounds at three different concentrations (25, 100, and 1000 nM) in this system. The 25 nM assay revealed that six compounds (20, 23, 24, 25, 29, and 22) possessed transcriptional activities considerably higher than that of compound 1 (bexarotene). Compounds 20, 23, 24, and 25 revealed transcriptional activity 2-fold higher than that of compound 1, while that of compounds 29 and 22 was increased 40−50% (Figure 6A). Conversely, compounds 31, 30, 27, and 26 exhibited dramatically reduced ability to induce RXR homodimerization and displayed activities of 0.6, 0.8, 3.4, and 11.3%, respectively, compared with compound 1. At 100 nM treatment (Figure 6B), four compounds (20, 23, 24, and 25) exhibited increased transcriptional activity in the range of 135−146% compared with compound 1. Additionally, compounds 29, 22, 28, and 21 had activity similar to that of compound 1. Analogous to the 25 nM treatment (Figure 6A), compounds 31 and 30 were not able to induce RXR homodimerization, but analogues 26 and 27 exhibited a significant increase in activity (5−16-fold) at the higher 100 nM analogue concentration (compare Figure 6A and Figure 6B). Furthermore, at 1000 nM treatment (Figure 6C), compounds 20, 23, 24, 25, and 26 exhibited activity greater than that of compound 1 by 35−58%. In contrast, even at this elevated concentration, compound 31 was not able to induce RXR homodimerization, while compound 30 had an activity of 17% compared with compound 1. Interestingly, compounds 29 and 22 exhibited the greatest transcriptional activity at 25 nM (140% and 150% of compound 1) and lower activity at 1000 nM (51−56% that of compound 1). These results indicate that novel analogues modeled after compound 1 can be generated that increase RXR homodimerization, presumably via increased binding to RXR (see Table 1). Furthermore, acidification of the molecule does not increase RXR homodimerization, as Table 1. EC50 Values Determined in the RXR Mammalian Two-Hybrid Assay

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Figure 7. Evaluation of RXR agonists via an RXRE-luciferase reporter-based assay in human colon cancer cells. HCT-116 human colon cancer cells were transfected with a human RXRα expression vector, an RXRE-luciferase reporter gene, and a renilla control plasmid. Cells were transfected for 24 h utilizing a liposome-mediated transfection protocol and then treated with either ethanol or DMSO vehicle or analogues at (A) 25 nM, (B) 100 nM, or(C) 1000 nM for 24 h. RXR-mediated transcription, as measured by luciferase output, was compared with that of the parent compound 1, which was set to 100%.

goal of this work was to develop specific RXR agonists, an index of cross-reactivity (CR) was generated by calculating the RARE:RXRE ratio, with compound 1 set to 1.0. Figure 8B reveals that RA, as expected, has a very high index of crossreactivity with a ratio of 9.5, indicating a preference to activate RAR over RXR. Conversely, compounds 25, 29, 22, and 28 have the lowest cross-reactivity index at 0.35−0.37, indicating that they preferentially bind and activate RXR 3-fold more efficiently than RAR (compared with the parent compound 1). Compounds 20, 26, and 27 have CR > 0.5. Taken together, this assay reveals that compounds 20−29 have a lower CR than compound 1, suggesting that they are more selective RXR agonists than the parent compound. Evaluation of SREBP Promoter Activation. One of the primary side effects of treatment with bexarotene (compound 1) is hypertriglyceridemia. Lipid metabolism is regulated in part by the sterol regulatory element-binding proteins (SREBPs), which are a group of transcription factors that regulate genes involved in lipid biosynthesis. Expression of SREBPs is regulated in part by the LXR−RXR heterodimer via LXRE control elements in the SREBP promoter. We tested the ability of compounds 20−31 to activate the SREBP promoter alone or in the presence of the LXR agonist TO901317 (TO). Figure 9A shows that compounds 26, 27, 29, 22, 28, and 21 have decreased transcriptional activity, ranging from 54 to 79% of the activity of compound 1, while compounds 20, 23, 24, and 25 have a similar activity profile relative to compound 1. Not surprisingly, compounds 31 and 30, which do not activate RXR (Figures 6 and 7), do not stimulate the SREBP promoter, and

homodimerization, DNA binding, and initiation of transcription via an RXRE. Conversely, compounds 31, 30, 26, and 27 are not as effective at initiating transcription from an RXRE as compound 1. As expected from the mammalian two-hybrid assay, compound 31 was unable to initiate transcription above the solvent control. At 100 nM, compounds 20, 23, 24, and 25 exhibited a 68−89% increase in transcriptional activity compared with compound 1 (Figure 7B). Compounds 29, 22, 28, and 21 showed slight increases in RXRE transcription of approximately 16−32%. At 1000 nM, only compounds 20, 23, 24, 25, and 26 exhibited increased activity, ranging from 20 to 43% (Figure 7C). Interestingly, compounds 27, 29, 22, 28, and 21 displayed similar RXRE activity as compound 1 despite having reduced activity in the mammalian two-hybrid system (compare Figure 6C and Figure 7C), suggesting that the specific DNA platform recognized by the RXR homodimer may play a role in agonist−receptor affinity/activity. Quantitation of RAR Agonist Activity. Since compound 1 is known to possess “residual” retinoic acid receptor (RAR) agonist activity, we evaluated the ability of compounds 20−31 to induce transcription via the retinoic acid response element (RARE) and RAR. Human embryonic kidney (HEK293) cells were transfected with human RARα and dosed with 100 nM of either all-trans-retinoic acid (RA), the natural ligand for RARα, compound 1, or analogues. Within this assay, compound 1 possessed 27% of the activity of RA (Figure 8A). Compound 20 displayed the greatest RARE activation at 26% of RA, while compound 29 showed the lowest RARE activity at 11%, which is indistinguishable from that of the ethanol control. Since one 8931

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fold preference for activating RXR/RXRE transcription versus SREBP (i.e., an LR of ≤0.50). Evaluation of RXR-Mediated Induction of CYP3A4. CYP3A4 is a member of the cytochrome P450 family of proteins and is responsible for the oxidation and clearance of approximately 50% of xenobiotics by the liver. It has been shown that compound 1 is sufficient to induce CYP3A4 expression.52 We tested compounds 20−31 for their ability to induce CYP3A4 mRNA expression using quantitative PCR (qPCR) in the liver carcinoma cell line HepG2. There was variable induction of CYP3A4 ranging from a 3-fold repression for compound 28 to a 2-fold induction for compounds 31 and 24, while compound 1 resulted in a 3.2-fold induction (Figure 10). In addition to compound 28, compound 21 also repressed CYP3A4 expression 2.3-fold. Compounds 23 and 29 did not alter CYP3A4 expression, while compounds 20, 25, 26, and 27 resulted in a modest increase (approximately 1.5-fold) in CYP3A4 expression. These results demonstrate that for one metabolic breakdown pathway, compounds 20−31 exert repressive effects that in vivo (depending on the totality of metabolism that occurs) could potentially translate into differential half-life profiles for these compounds relative to 1, as previously observed by our group.46 Inhibition of Proliferation in CTCL Cells. Since compound 1 is used clinically to treat cutaneous T-cell lymphoma (CTCL), compounds 20−31 were evaluated for their ability to inhibit growth of the CTCL cell line Hut78. Figure 11 indicates that none of the compounds tested were as efficient at inhibiting Hut78 growth as compound 1. However, compounds 20, 23, and 24 were able to inhibit growth >85% as efficiently as compound 1. Furthermore, compounds 25, 29, and 21 also maintained 55−80% of the antiproliferative capacity of the parent compound 1. There was a substantial decrease in the antiproliferative effect for compounds 26, 27, 22, and 28, which were only 35−51% as effective as compound 1. Inhibition of HDAC1 Expression in CTCL Cells. Since none of the novel analogues tested were as effective at inhibiting cell growth of Hut78 cells, we examined the ability of a subset of analogues (20 and 25) to inhibit histone deacetylase-1 (HDAC1) expression. HDAC inhibitors have been in development for several years, with four receiving FDA approval, including vorinostat for treatment of CTCL.53 Figure 12 shows that compound 1 was able to suppress HDAC1 expression by 10%, while compound 25 showed a 28% reduction. Surprisingly, compound 20 did not alter HDAC1 expression. These data suggest that although compound 25 did not inhibit growth as effectively as compound 1 in the proliferation assay (Figure 11), it may be more effective at inducing toxicity or apoptosis by altering gene expression and protein function compared with compound 1. Mutagenesis. Further determining the suitability of the compounds, we undertook a screen of the compounds to test for their ability to mutate DNA, a very undesirable trait in any potential pharmaceutical. We assayed the compounds as in previous work39,45 and determined that none of the compounds are mutagenic in this assay.

Figure 8. Assessment of RXR agonists via an RARE-luciferase reporter-based assay in human kidney cells. (A) Human embryonic kidney (HEK293) cells were cotransfected with expression vectors for hRXRα, hRAR, a RARE-luciferase reporter gene, and a renilla control plasmid for 24 h utilizing a liposome-mediated transfection protocol. Cells were treated with analogue or all-trans-retinoic acid (RA) at 100 nM for 24 h. The RARE activity for RA was set to 100%. (B) The “index of cross-reactivity” was determined from the RARE:RXRE ratio activity, with compound 1 set to 1.0.

the activity of these analogues is similar to that of the ethanol control. Furthermore, in the presence of analogue and TO (activated LXR), only compounds 26, 27, 28, and 21 show reduced SREBP promoter activation compared with compound 1 and TO. Compound 26 + TO shows a reduction of 33%, and compound 27 displays a 29% reduction. Although compounds 20, 23, 24, and 25 have similar transcriptional activity as compound 1 alone, there is a 13−17% decrease when they are combined with TO. The lipid risk assessment (LR) was generated by calculating the SREBP:RXRE activity ratio and is a measure of the potential risk of SREBP-driven hypertriglyceridemia associated with patient treatment of compound 1. Compounds 26, 29, 22, and 28 show LRs ranging from 0.48 to 0.65 (Figure 9B), with compounds 29, 22, 28, and 21 possessing both an increase in RXRE transcriptional activity (Figure 7B) and a decrease in SREBP transcription (Figure 9A) compared with compound 1. These results suggest that it is possible to generate analogues of compound 1 with increased RXR activity, while potentially decreasing adverse side effects of hypertriglyceridemia. Conversely, compound 27 possesses both a decrease in RXR/RXRE transcriptional assays (Figure 7B) and a decrease in SREBP promoter activity (Figure 9A). Furthermore, the LRs for compounds 20, 23, 24, and 25 range from 0.49 to 0.51 as a result of increased transcriptional activity in the RXRE assay of 68−89% (Figure 7B), while similar SREBP transcriptional activity as for compound 1 is maintained (Figure 9A). In summary, the results for compounds 20, 23, 24, 25, and 28 indicate that these compounds display at least a 2-



CONCLUSIONS Bexarotene (1) possesses FDA approval for cutaneous T-cell lymphoma (CTCL) and has been examined in a plethora of other cancer types. Recent studies also suggest that 1 may be a promising candidate to treat Alzheimer’s disease (AD).29,54 The current study was interested in factors that may mitigate the 8932

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Figure 9. Evaluation of RXR agonists to potentiate SREBP promoter activation in the absence and presence of LXR ligand TO901317 (TO). (A) HCT-116 human colon cancer cells were transfected with an expression vector for human LXRα, an SREBP-promoter-luciferase reporter gene, and a renilla control plasmid. Cells were transfected for 24 h utilizing a liposome-mediated transfection protocol and then treated with ethanol or DMSO vehicle or the indicated compound at 100 nM either alone or in combination with 100 nM TO. SREBP-promoter-directed activity was compared with that of compound 1, which was set to 100%. (B) The “lipid risk assessment” was determined by the SREBP:RXRE ratio, with compound 1 set to 1.0.

Figure 10. Assessment of RXR agonists to induce CYP3A4 expression. Human hepatocellular carcinoma (HepG2) cells were treated for 24 h with ethanol or DMSO vehicle or the indicated compound at 100 nM. Total RNA was extracted, and 0.5 μg of DNase-treated sample was used for cDNA synthesis and subsequent qPCR. Data analysis was performed using the comparative ΔΔCt method as the means of relative quantitation, normalized to an endogenous reference (GAPDH) and relative to a calibrator (normalized Ct value from vehicle-treated cells) and expressed as 2−ΔΔCt. Ethanol and DMSO are set to 1.0.

the same effect in patients, and this may have the potential to reduce dose-dependent side effects. We addressed the primary concern about rexinoids’ tendency to raise lipids by assessing this panel of analogues in an SREBP assay. Interestingly, compounds 21, 22, 28, and 29 all exhibit greater RXRE activation and reduced SREBP promotion versus 1. Furthermore, given the recent development of vorinostat and romidepsin histone deacetylase inhibitors (HDACi) and FDA approval of these HDACi for the treatment of CTCL, we found that compound 25 exhibited greater suppression of HDAC1 than 1. Moreover, several of the rexinoids 20−31 also demonstrated similar antiproliferative effects in Hut78 cells as well as lower induction of CYP3A4 compared with 1,

side effect profile of 1, since bexarotene appears to effectively treat many CTCL patients but also has the potential to activate RXR heterodimer pathways and thereby induce hyperlipidemia and hypothyroidism. There is compelling motivation to explore novel RXR ligands for therapeutic treatment, since new rexinoids may possess unique activities and reduced side effect profiles, and we have previously demonstrated39,45 a number of novel rexinoids with different PK characteristics and differential ability to modulate RXR-controlled pathways.46 The current study produced nine rexinoids, five of which were determined to possess lower EC50 values than 1. More potent rexinoids, possessing lower EC50 values, may be able to translate to clinical treatments, permitting lower dose regimens to achieve 8933

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Figure 11. Probing of compound 1 and selected analogues for inhibition of cell growth utilizing an MTS assay in CTCL. Human cutaneous T-cell lymphoma (Hut78) cells were plated at 7500 cells per well and immediately dosed with either ethanol or DMSO vehicle or the indicated analogue at 200 nM for 72 h. RXR-mediated inhibition of growth was compared to that for compound 1, set to 100%. The data represent averages of three independent experiments. glutamine + sodium pyruvate (Mediatech, Manassas, VA) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), supplemented with 100 μg/mL streptomycin and 100 unit/mL penicillin. (Gibco, Gaithersberg, MD). Cells were cotransfected using a human RXR binding domain (BD) vector (bait), a human RXR activation domain (AD) vector (prey), pFR-Luc, and renilla control plasmid. Transfection was conducted using 1.25 μL of Polyjet (Signagen, Gaithersberg, MD) per well and allowed to incubate for 24 h. The cells were then treated with ethanol or DMSO vehicle (0.1%), compound 1, or the indicated analogue at a final concentration of 25, 100, or 1000 nM and incubated for 24 h. The rexinoid activity was measured by luciferase output utilizing a dual-luciferase report assay system according to the manufacturer’s instructions (Promega, Madison, WI) in a Sirus luminometer (Berthold Detection Systems, Pforzheim, Germany). Three independent assays were conducted with triplicate samples for each treatment group. The value for compound 1 was set to 100%. EC50 Determination. The EC50 values were determined from full dose−response curves for compound 1 and compounds 20−29 ranging from 0.1 to 2000 nM. EC50 curves for 30 and 31 were determined from dose−response curves ranging from 1 to 80 μM. HCT-116 cells were plated, transfected, and treated as above using the mammalian two-hybrid assay protocol. Cells were treated with ethanol or DMSO vehicle (0.1%) or analogue 20−29 (final concentration of 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, or 2000 nM) for 24 h. Alternatively, dose−response curves for compounds 30 and 31 were generated using a final concentration of 1, 2.5, 5, 10, 20, 40, or 80 μM. The amount of rexinoid activity at each concentration was measured using the same luciferase assay as described above. Dose− response curves were generated using four-parameter analysis utilizing Prism Software (GraphPad Inc., La Jolla, CA). Three independent assays were conducted with triplicate samples for each treatment group. RXRE-Mediated Transcription Assay. HCT-116 cells were plated at 80 000 cells per well in a 24-well plate and maintained as described above. The cells were cotransfected using 250 ng of RXREluciferase reporter gene (RXRE from the naturally occurring responsive element in rat cellular retinol binding protein II gene: 5′AAAATGAACTGTGACCTGTGACCTGTGACCTGTGAC-3′, in which DR1-responsive elements are underlined), 25 ng of pSG5human RXRα, and 20 ng of renilla control plasmid using 1.25 μL of Polyjet for 24 h. The cells were treated with ethanol or DMSO vehicle (0.1%) or analogue (final concentration of 25, 100, or 1000 nM) for 24 h. The rexinoid activity was measured by luciferase output as described above. Three independent assays were conducted with triplicate samples for each treatment group. The value for compound 1 was set to 100%. RARE-Mediated Transcription Assay. Human embryonic kidney (HEK293) cells were plated at 60 000 cells per well in a 24-well plate and maintained as described above. After 24 h, the cells were transfected with 250 ng of pTK-DR5(X2)-Luc, 25 ng of pCMX-

Figure 12. Ability of RXR agonists to repress HDAC-1 expression. Human cutaneous T-cell lymphoma (Hut78) cells were treated for 48 h with ethanol or the indicated compound at 100 nM. A 0.5 μg portion of DNase-treated RNA was used for cDNA synthesis and subsequent qPCR. Data analysis was performed using the comparative ΔΔCt method as the means of relative quantitation, normalized to an endogenous reference (GAPDH) and relative to a calibrator (normalized Ct value from vehicle-treated cells) and expressed as 2−ΔΔCt. Ethanol is set to 1.0. * indicates p < 0.05 vs compound 1 utilizing an unpaired homoscedastic Student’s t test.

suggesting that some of the novel rexinoids may perhaps possess modified half-life profiles in comparison with 1. Taken together, these results indicate that a number of our novel RXR agonists in this study exhibit greater on-target potency, similar efficacy at inhibition of CTCL proliferation, and a lower likelihood to induce side effects than 1.



EXPERIMENTAL SECTION

Ligand Docking and Scoring. Docking studies were carried out for bexarotene and ligands 20−31. The ligands were prepared by Avogadro55 following an energy minimization with the Universal Force Field.56 The charge distributions on ligands were assigned by Autodock57 and OpenBabel 2.3.0.58 Prepared ligands were docked to a group of target proteins, which included human RXRα (PDB entries 1MVC,50 1MZN,50 1RDT,59 2P1T,8 2P1U,8 2ZXZ,60 2ZY0,60 3DZY,61 3DZU,61 3E00,61 3FUG,43 3H0A,62 3PCU,63 and 4K6I62) and RXRβ (PDB entry 1H9U64). Docking was performed using the Autodock 4.2 software.57 As in our previous studies,39,45 protein residues Arg 316 and Ile 268 (residue numbering as in 1MVC) were treated as flexible. Docking used the Lamarckian genetic algorithm with 25 000 000 energy evaluations per dock and a total of 250 docks per compound. Calculated binding free energies were used to score the compounds. Mammalian Two-Hybrid Assay. HCT-116 colorectal carcinoma cells were plated overnight at 80 000 cells per well in a 24-well plate and maintained in Dulbecco’s modified Eagle’s medium (DMEM) + 8934

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human RARα, and 20 ng of renilla utilizing 1.25 μL of Polyjet per well for 24 h. The sequence of the double DR5 RARE is 5′AAAGGTCACCGAAAGGTCACCATCCCGGGAGGTCACCGAAAGGTCACC-3′ (in which DR5-responsive elements are underlined). The cells were treated with ethanol or DMSO vehicle (0.1%), all-transretinoic acid (RA, the ligand for RAR), or the indicated rexinoid at a final concentration of 100 nM. After 24 h of treatment, the retinoid activity was measured as described above. The activity of compound 1 or analogue divided by the activity of RA (expressed as a percentage) represents the RAR agonist activity. Three independent assays were conducted with triplicate samples for each treatment group. The value for RA was set to 100%. The index of cross-reactivity was calculated by dividing the RARE activity by the 100 nM RXRE activity with the RARE:RXRE ratio for compound 1 set to 1.0. Sterol Regulatory Element-Binding Protein (SREBP)-Mediated Transcription Assay. HCT116 colorectal carcinoma cells were plated at 80 000 cells per well and maintained as described above. After 24 h, the cells were cotransfected with 100 ng of pBP1c(6500)-Luc, which contains approximately 6500 base pairs of the flanking DNA from the murine SREBP-1c natural promoter, along with 10 ng of pCMX-hLXRα and 20 ng of renilla control plasmid using 1.25 μL of Polyjet for 24 h. The cells were treated with 100 nM compound 1 or analogue alone or with 100 nM LXR agonist TO901317. After 24 h, the amount of SREBP promoter activity was measured by luciferase output as described above. Three independent assays were conducted with triplicate samples for each treatment group. The value for compound 1 was set to 100%. The lipid risk assessment was calculated by dividing the SREBP activity by 100 nM RXRE, with the SREBP:RXRE ratio for compound 1 set to 1.0. Proliferation Assay. Human T-cell lymphoma (Hut78) cells were maintained in Roswell Park Memorial Institute (RPMI) medium (Mediatech) containing 10% FBS (Atlanta Biologicals), supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin (Gibco) at 37 °C, 5% CO2. Cells were plated at 7500 cells per well in a 96-well plate and immediately dosed with either medium alone, ethanol, DMSO, or 200 nM compound 1 or analogue in a total volume of 100 μL. After 72 h, 20 μL of CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega) was added to each well, and the plate was incubated at 37 °C according to the manufacturer’s recommendations. MTS assays were quantified using a BioTek ELx808 96-well plate reader (BioTek, Winooski, VT) at A490nm. Percent inhibition was calculated using the value for the medium control set to 0.0. Quantitative Real-Time PCR. Human hepatocellular carcinoma (HepG2) cells were maintained in DMEM + glutamine + sodium pyruvate (Mediatech) containing 10% FBS (Atlanta Biologicals), supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin (Gibco). Cells were plated at 400 000 cells per well in a sixwell plate. After 16 h, the medium was replaced with DMEM containing 1% FBS for 24 h. The cells were dosed in DMEM/1% FBS containing ethanol, DMSO, compound 1, or analogue for 24 h. Total RNA was isolated from each plate or well using an Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The RNA obtained was quantified using A260/280 spectrophotometry. Hut78 cells were maintained in RPMI medium as described above. Cells were harvested by centrifugation for 10 min at 400 rcf at room temperature and then resuspended in RPMI medium supplemented with 1% FBS, 100 μg/mL streptomycin, and 100 units/mL penicillin. Cells were plated at 750 000 cells per well in a six-well plate and immediately dosed with ethanol, DMSO, compound 1, or analogue at a final concentration of 100 nM for 48 h. Total RNA was extracted as described above. DNase-treated total RNA (0.5 μg) was reverse-transcribed via the use of the iScript cDNA Synthesis kit (Bio-Rad) to prepare 20 μL of first-strand cDNA. Real-time PCR reactions were prepared by adding 1.5 μL of cDNA to 5 μL of FastStart Universal SYBR Green Master Mix + Rox (Roche Applied Science, Indianapolis, IN) and primers for a total volume of 10 μL. Reactions were performed in 96-well plates in a BioRad CFX96 thermal cycler using a 40 cycle profile. Data analysis

was performed using the comparative ΔΔCt method as the means of relative quantitation, normalized to an endogenous reference (GAPDH) and relative to a calibrator (normalized Ct value from vehicle-treated cells) and expressed as 2−ΔΔCt according to Applied Biosystems’ User Bulletin 2, revision B, “Relative Quantitation of Gene Expression”. The primers utilized during PCR experimentation are as follows: GAPDH forward, 5′-ACAACTTTGGTATCGTGAAGGAC3′; GAPDH reverse, 5′-CAGGGATGATGTTCTGGAGAGC-3′; HDAC1 forward, 5′-GAGATGACCAAGTACCACAGC-3′; HDAC1 reverse, 5′-TGACAGAACTCAAACAGGCC-3′; CYP3A4 forward, 5′TCAGCCTGGTGCTCCTCTATCTAT-3′; CYP3A4 reverse, 5′AAGCCCTTATGGTAGGACAAAATATTT-3′. Mutagenesis Assay. All of the compounds were tested for their ability to mutate DNA in an EPA-approved Saccharomyces cerevisiae cell-based system as in our previous work.39,45 D7 yeast cells65 were incubated with increasing concentrations of each compound solubilized in DMSO (highest concentration 0.1 mg/mL) and then plated on yeast extract, peptone, dextrose medium or on synthetic medium lacking isoleucine or tryptophan to check for forward and reverse mutations.66 Instrumentation. A 400 MHz Bruker spectrometer was used to acquire 1H and 13C NMR spectra. For 1H NMR spectra, chemical shifts (δ) are listed in parts per million against residual nondeuterated solvent peaks in a given deuterated solvent (e.g., CHCl3 in CDCl3) as an internal reference. Coupling constants (J) are reported in hertz, and the following abbreviations for splittings are used: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; br, broad. All of the 13C NMR spectra were acquired on a Bruker instrument at 100.6 MHz. Chemical shifts (δ) are listed in parts per million against deuterated solvent carbon peaks as an internal reference. Highresolution mass spectra were recorded using either a JEOL GCmate(2004) or JEOL LCmate(2002) high-resolution mass spectrometer or an ABI Mariner (1999) ESI-TOF mass spectrometer. General Procedures. Removal of volatile solvents was performed under reduced pressure using a Büchi rotary evaporator and is described hereafter as “concentration in vacuo”. Thin-layer chromatography was conducted on precoated (0.25 mm thick) silica gel plates with 60F-254 indicator (Merck). Column chromatography was conducted using 230−400 mesh silica gel (E. Merck reagent silica gel 60). All of the tested compounds were analyzed for purity via combustion analysis by Columbia Analytical Services (formerly Desert Analytics, Tucson, AZ) and were found to be >95% pure. NEt-TMN (20).47a To a 100 mL round-bottom flask equipped with a stir bar and charged with methyl 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8tetrahydronaphthalen-2-yl)amino)nicotinate (0.3674 g, 1.002 mmol) suspended in methanol (3.5 mL) was added a solution of potassium hydroxide (0.1767 g, 3.15 mmol) in water (0.22 mL). This reaction mixture was stirred at reflux in an oil bath at 87 °C for 1 h, cooled to room temperature, and acidified with 20% HCl (52 mL). The crude white precipitate was filtered and washed with cold water to provide crude 20 (0.3238 g, 91%), and this crude material was purified by column chromatography (25 mL SiO2, 30% ethyl acetate/hexanes to pure ethyl acetate to 2% methanol/ethyl acetate) to give 20 (0.2677 g, 76%) as a white crystalline solid, mp 230−232 °C (lit. 235.7−237.7 °C): 1H NMR (400 MHz, DMSO-d6) δ 12.44 (br s, 1H), 8.66 (dd, J = 2.4, 0.4, 1H), 7.78 (dd, J = 8.8, 2.4, 1H), 7.43 (d, J = 8.0, 1H), 7.18 (d, J = 2.0, 1H), 7.01 (dd, J = 8.4, 2.4, 1H), 6.19 (dd, J = 9.2, 0.4, 1H), 3.94 (q, J = 6.8, 2H), 1.66 (s, 4H), 1.26 (s, 6H), 1.22 (s, 6H), 1.22 (t, J = 6.8, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 166.6, 159.7, 150.5, 146.5, 143.1, 140.7, 137.6, 128.1, 125.4, 124.8, 114.7, 106.7, 44.7, 34.5. 34.4, 34.0, 33.8, 31.5, 31.4, 12.7; IR (neat) 2925, 1666, 1592, 1409, 1274 cm−1; ESI-MS (M − H)− calcd for C22H27N2O2 351.2072, found 351.2073. Anal. Calcd for C22H28N2O2: C, 74.97; H, 8.01; N, 7.95. Found: C, 74.74; H, 8.38; N, 7.56. 4-(Ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen2-yl)amino)benzoic Acid (21). Compound 21 was synthesized following the same general procedure as for 20, which gave pure 21 as a white crystalline solid, mp 247.8−250.6 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.14 (br s, 1H), 7.69 (d, J = 9.2, 2H), 7.38 (d, J = 8.4, 1H), 7.12 (d, J = 2.0, 1H), 6.95 (dd, J = 8.4, 2.4, 1H), 6.64 (d, J = 8.8, 8935

DOI: 10.1021/acs.jmedchem.6b00812 J. Med. Chem. 2016, 59, 8924−8940

Journal of Medicinal Chemistry

Article

2H), 3.72 (q, J = 7.2, 1H), 1.65 (s, 4H), 1.26 (s, 6H), 1.21 (s, 6H), 1.13 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 167.2, 151.1, 146.1, 142.6, 141.9, 130.9, 127.9, 124.6, 124.1, 118.2, 112.6, 34.5, 34.4, 34.0, 33.7, 31.5, 31.4, 12.1; IR (neat) 2955, 1661, 1594, 1270, 1180 cm−1; ESI-MS (M − H)− calcd for C23H28NO2 350.2120, found 350.2122. Anal. Calcd for C23H29NO2: C, 78.59; H, 8.32; N, 3.99. Found: C, 78.30; H, 8.70; N, 3.87. 4-(Ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoic Acid (22). Compound 22 was synthesized following the same general procedure as for 20, which gave pure 22 as a white crystalline solid, mp 252.4−256.8 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.06 (br s, 1H), 7.68 (d, J = 9.2, 2H), 7.28 (s, 1H), 7.02 (s, 1H), 6.40 (d, J = 8.8, 2H), 3.62 (br s, 2H), 1.97 (s, 3H), 1.63 (s, 4H), 1.25 (s, 6H), 1.19 (s, 6H), 1.14 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 167.3, 151.1, 144.0, 143.2, 140.7, 132.8, 131.1, 129.1, 126.6, 117.3, 110.7, 45.4, 34.6, 34.4, 33.7, 33.6, 31.6, 31.5, 17.0, 12.2; IR (neat) 2957, 1665, 1597, 1274, 1176 cm−1; ESI-MS (M − H)− calcd for C24H30NO2 364.2277, found 364.2268. Anal. Calcd for C24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.87; H, 8.91; N, 3.76. 6-(Ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic Acid (23). The same methodology as was used to synthesize 20 gave pure 23 as a white crystalline solid, mp 250.2−251.0 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.42 (br s, 1H), 8.66 (d, J = 2.0, 1H), 7.78 (dd, J = 8.8, 2.0, 1H), 7.32 (s, 1H), 7.07 (s, 1H), 5.89 (br s, 1H), 4.19−4.05 (m, 1H), 3.65−3.55 (m, 1H), 1.98 (s, 3H), 1.64 (s, 4H), 1.26 (d, J = 6.4, 6H), 1.20 (s, 6H), 1.13 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 166.6, 159.5, 150.7, 144.1, 143.8, 139.0, 137.9, 132.5, 129.2, 126.5, 114.4, 105.9, 44.0, 34.5, 34.4, 33.7, 31.6, 31.5, 16.9, 12.7; IR (neat) 2960, 1669, 1595, 1509, 1412, 1266 cm−1; ESI-MS (M − H)− calcd for C23H29N2O2 365.2229, found 365.2235. Anal. Calcd for C23H30N2O2: C, 75.37; H, 8.25; N, 7.64. Found: C, 75.33; H, 8.46; N, 7.56. 2-(Ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen2-yl)amino)pyrimidine-5-carboxylic Acid (24). The same methodology as was used to synthesize 20 gave 24 (0.3668 g, 97%) as a white crystalline solid, mp 249.5−250.6 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.62 (br s, 1H), 8.37 (s, 1H), 7.35 (d, J = 8.4, 1H), 7.17 (d, J = 2.4, 1H), 7.00 (dd, J = 8.4, 2.0, 1H), 3.99 (q, J = 7.2, 2H), 1.67 (s, 4H), 1.27 (s, 6H), 1.24 (s, 6H), 1.16 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 165.1, 161.9, 159.1, 15.2, 142.3, 140.2, 126.8, 124.7, 124.6, 113.5, 45.3, 34.4, 34.4, 33.7, 33.5, 31.4, 31.3, 12.3; IR (neat) 2962, 1664, 1586, 1515, 1426, 1278 cm−1; ESI-MS (M − H)− calcd for C21H26N3O2 352.2025, found 352.2024. Anal. Calcd for C21H27N3O2: C, 71.36; H, 7.70; N, 11.89. Found: C, 71.40; H, 7.98; N, 11.79. 2-(Ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrimidine-5-carboxylic Acid (25). The same methodology as was used to synthesize 20 gave pure 25 as a white crystalline solid, mp 231.9−233.1 °C: 1H NMR (400 MHz, DMSOd6) δ 12.60 (br s, 1H), 8.74 (br s, 2H), 7.23 (s, 1H), 7.05 (s, 1H), 4.04 (sextet, J = 7.2, 1H), 3.76 (sextet, J = 7.2, 1H), 1.97 (s, 3H), 1.65 (s, 4H), 1.29 (s, 3H), 1.26 (s, 3H), 1.23 (s, 3H), 1.20 (s, 3H), 1.16 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 165.2, 161.7, 159.3, 143.0, 142.9, 139.1, 132.0, 128.2, 125.4, 113.2, 45.0, 34.5, 34.4, 33.4, 33.3, 31.5, 31.4, 31.2, 16.9, 12.3; IR (neat) 2952, 1663, 1591, 1508, 1426, 1281 cm−1; ESI-MS (M − H)− calcd for C22H28N3O2 366.2181, found 366.2185. Anal. Calcd for C22H29N3O2: C, 71.90; H, 7.95; N, 11.43. Found: C, 71.81; H, 8.23; N, 11.31. 5-(Ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen2-yl)amino)pyrazine-2-carboxylic Acid (26). The same methodology as was used to synthesize 20 gave 26 (0.2516 g, 71%) as a white crystalline solid, mp 213.1−214.4 °C: 1H NMR (400 MHz, DMSOd6) δ 12.59 (br s, 1H), 8.70 (d, J = 1.6, 1H), 7.61 (d, J = 1.2, 1H), 7.47 (d, J = 8.0, 1H), 7.29 (d, J = 2.4, 1H), 7.10 (dd, J = 8.4, 2.4, 1H), 3.94 (q, J = 7.1, 2H), 1.66 (s, 4H), 1.27 (s, 6H), 1.23 (s, 6H), 1.14 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 165.4, 154.4, 146.7, 144.6, 143.8, 139.2, 130.8, 130.7, 128.4, 125.2, 124.4, 44.9, 34.4, 34.4, 34.1, 33.8, 31.5, 31.4, 12.2; IR (neat) 2931, 1671, 1556, 1416, 1276 cm−1; ESI-MS (M − H)− calcd for C21H26N3O2 352.2025, found

352.2020. Anal. Calcd for C21H27N3O2: C, 71.36; H, 7.70; N, 11.89. Found: C, 70.43; H, 7.64; N, 11.61. 5-(Ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)pyrazine-2-carboxylic Acid (27). The same methodology as was used to synthesize 20 gave pure 27 as a white crystalline solid, mp 203.6−205.1 °C: 1H NMR (400 MHz, DMSOd6) δ 12.69 (br s, 1H), 8.70 (s, 1H), 7.36 (br s, 2H), 7.15 (s, 1H), 4.15−4.05 (m, 1H), 3.59 (sextet, J = 7.2, 1H), 2.03 (s, 3H), 1.65 (s, 4H), 1.29 (s, 3H), 1.26 (s, 3H), 1.22 (s, 3H), 1.20 (s, 3H), 1.15 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 165.4, 154.2, 144.8, 144.4, 144.3, 137.6, 132.3, 130.8, 130.0, 129.5, 126.4, 44.1, 34.5, 34.4, 33.7, 31.6. 31.5, 16.8, 12.1; IR (neat) 2959, 1671, 1557, 1524, 1417, 1286 cm−1; ESI-MS (M − H)− calcd for C22H28N3O2 366.2181, found 366.2179. Anal. Calcd for C22H29N3O2: C, 71.90; H, 7.95; N, 11.43. Found: C, 71.62; H, 8.21; N, 11.19. 4-(Ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen2-yl)amino)-2-fluorobenzoic Acid (28). The same methodology as was used to synthesize 20 gave pure 28 as a white crystalline solid, mp 252.4−256.8 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.35 (br s, 1H), 7.62 (t, J = 9.2, 1H), 7.41 (d, J = 8.4, 1H), 7.14 (d, J = 2.4, 1H), 6.97 (dd, J = 8.4, 2.4, 2H), 6.38 (dd, J = 9.2, 2.4, 1H), 6.32 (dd, J = 14.8, 2.4, 1H), 3.71 (q, J = 7.2, 2H), 1.65 (s, 4H), 1.26 (s, 6H), 1.21 (s, 6H), 1.12 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 164.9, 164.8, 164.5, 161.9, 153.0, 152.9, 146.4, 142.7, 141.9, 133.2, 128.1, 125.1, 124.5, 108.6, 105.7, 105.6, 99.7, 99.4, 46.3, 34.5, 34.4, 34.0, 33.7, 31.5, 31.4, 12.0; IR (neat) 2964, 1668, 1617, 1282 cm−1; ESI-MS (M − H)− calcd for C23H27FNO2 368.2026, found 368.2043. Anal. Calcd for C23H28FNO2: C, 74.77; H, 7.64; N, 3.79; F, 5.14. Found: C, 74.42; H, 8.00; N, 3.64; F, 4.1. Methyl 2-Fluoro-4-((3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)benzoate (65). The same methodology as was used to synthesize 43 gave 65 (1.9009 g, 65%) as a crystalline solid, mp 159.7−165.1 °C: 1H NMR (400 MHz, CDCl3) δ 7.79 (t, J = 8.8, 1H), 7.17 (d, J = 3.6, 1H), 6.51 (dd, J = 8.8, 2.0, 1H), 6.38 (dd, J = 13.6, 2.0, 1H), 5.74 (br s, 1H), 3.87 (s, 3H), 2.17 (s, 3H), 1.68 (s, 4H), 1.29 (s, 6H), 1.24 (s, 6H); 13C NMR (100.6 MHz, CDCl3) δ 165.3, 165.0, 164.9, 162.7, 151.9, 151.8, 143.9, 142.6, 135.2, 133.6, 133.5, 129.9, 129.1, 122.5, 109.6, 109.5, 107.5, 107.4, 100.9, 100.6, 51.7, 35.0. 34.9, 34.0, 33.9, 31.8; IR (neat) 3346, 2922, 1698, 1606, 1264 cm−1; ESI-MS (M + Na)+ calcd for C23H28FNO2Na 392.2002, found 392.2003. 4-(Ethyl(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)-2-fluorobenzoic Acid (29). The same methodology as was used to synthesize 20 gave pure 29 as a white crystalline solid, mp 241.4−243.8 °C: 1H NMR (400 MHz, DMSO-d6) δ 12.29 (br s, 1H), 7.62 (t, J = 8.8, 1H), 7.31 (s, 1H), 7.03 (s, 1H), 6.19−6.11 (m, 2H), 3.61 (br s, 2H), 1.98 (s, 3H), 1.63 (s, 4H), 1.26 (s, 6H), 1.20 (s, 6H), 1.13 (t, J = 7.2, 3H); 13C NMR (100.6 MHz, DMSO-d6) δ 164.9, 164.9, 164.7, 162.2, 153.0, 152.9, 144.1, 143.7, 140.0, 133.4, 132.6, 129.3, 126.5, 107.3, 105.1, 105.0, 98.3, 98.0, 45.6, 34.5, 34.4, 33.7, 31.6, 31.5, 16.9, 12.1; IR (neat) 2922, 1670, 1606, 1285 cm−1; ESI-MS (M − H)− calcd for C24H29FNO2 382.2182, found 382.2170. Anal. Calcd for C24H30FNO2: C, 75.16; H, 7.88; N, 3.65; F, 4.95. Found: C, 75.28; H, 8.45; N, 3.61; F, 4.3. 4-(1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen2-yl)vinyl)benzenesulfonic Acid (30). To a 100 mL round-bottom flask equipped with a magnetic stir bar and charged with 71 (0.2046 g, 0.464 mmol) in methanol (3.0 mL) was added a solution of potassium hydroxide (0.0902 g, 1.61 mmol) in water (0.18 mL). The flask was fitted with a water condenser and heated to reflux in an oil bath at 85 °C for 2 h. The reaction mixture was cooled to room temperature, and 20% HCl (30 mL) was added. The resulting solution was extracted with ethyl acetate (50 mL, twice), and the combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified by column chromatography (25 mL of SiO2, 10% methanol/ ethyl acetate) to give 30 (0.1436 g, 80%) as a white crystalline solid, decomp. >280 °C: 1H NMR (400 MHz, DMSO-d6) δ 7.55 (d, J = 8.4, 2H), 7.18 (d, J = 8.4, 2H), 7.13 (s, 1H), 7.05 (s, 1H), 5.79 (d, J = 1.2, 1H), 5.14 (d, J = 1.2, 1H), 1.91 (s, 3H), 1.64 (s, 4H), 1.26 (s, 6H), 1.22 (s, 6H); 13C NMR (100.6 MHz, DMSO-d6) δ 148.5, 147.4, 143.5, 8936

DOI: 10.1021/acs.jmedchem.6b00812 J. Med. Chem. 2016, 59, 8924−8940

Journal of Medicinal Chemistry

Article

141.7, 140.2, 138.2, 132.1, 127.7, 127.2, 125.7, 125.4, 115.4, 34.7, 34.6, 33.6, 33.4, 31.7, 31.6, 19.5; IR (neat) 2961, 1455, 1179, 1043, 1008, 845, 669 cm−1; ESI-MS (M − H)− calcd for C23H27SO3 383.1681, found 383.1667. Anal. Calcd for C23H28O4S·2H2O: C, 65.68; H, 7.67; S, 7.62. Found: C, 63.94; H, 7.35; S, 7.15. 4-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalene-2carbonyl)benzenesulfonic Acid (31). Acetone (2.0 mL) was added to a 20 dram vial charged with 69 (0.5621 g, 1.39 mmol), and the vial was gently heated until the solution was homogeneous. To this solution of 69 in acetone was added a solution of potassium hydroxide (0.213 g, 3.80 mmol) in water (0.25 mL). The reaction mixture was stirred and gently warmed to keep the solution homogeneous for 30 min, at which point the reaction was quenched with 20% HCl (20 mL) and the resulting mixture was extracted with ethyl acetate (50 mL, thrice). The combined organic layers were washed with water (50 mL) and then brine (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a crude product that was purified by column chromatography (25 mL SiO2, 10% methanol/ethyl acetate) to give 31 (0.300 g, 56%) as a white crystalline solid, decomp. >200 °C: 1H NMR (400 MHz, DMSO-d6) δ 7.75 (dd, J = 6.4, 1.6, 2H), 7.66 (dd, J = 6.4, 1.6, 2H), 7.30 (s, 1H), 7.21 (s, 1H), 2.21 (s, 3H), 1.65 (s, 4H), 1.28 (s, 6H), 1.18 (s, 6H); 13C NMR (100.6 MHz, DMSO-d6) δ 197.1, 152.3, 147.1, 141.4, 137.3, 135.3, 133.2, 129.4, 128.9, 127.0, 125.8, 34.4, 34.3, 33.9, 33.5, 31.4, 31.3, 19.3; IR (neat) 2925, 1673, 1191, 1123, 1038 cm−1; ESI-MS (M − H)− calcd for C22H25SO4 385.1474, found 385.1472. Anal. Calcd for C22H26O4S·2H2O: C, 62.54; H, 7.16; S, 7.59. Found: C, 61.75; H, 6.76; S, 7.25.



described in this work have been submitted on behalf of the Arizona Board of Regents.



ABBREVIATIONS: CR, index of cross-reactivity of RAR and RXR; CTCL, cutaneous T-cell lymphoma; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; HRE, hormoneresponsive element; LBD, ligand-binding domain; LXR, liver X receptor; LR, lipid risk assessment index; NaBu, sodium butyrate; NR, nuclear receptor; PPAR, peroxisome proliferatoractivated receptor; POC, proof of concept; RAR, retinoic acid receptor; RXR, retinoid X receptor; RXRE, RXR-responsive element; SNuRM, specific nuclear receptor modulator; SREBP, sterol regulatory element-binding protein; TR, thyroid hormone receptor; VDR, vitamin D receptor



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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00812. CCDC numbers 1480108−1480113 contain the supplementary crystallographic data for this work. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. Full experimental details for the synthesis of 20−31 and all synthetic intermediates (33, 35, 36, 38, 39, 41−44, 46−48, 50, 51, 53, 54, 56, 57, 59, 60, 62−66, and 68− 71); competitive binding of 30 and 31 to RXR versus 1; 1 H and 13C NMR spectra; and X-ray data for compounds 20, 21, 26, 28, 44, and 60 (PDF) SMILES strings and HCT-116 EC50 data (CSV)



AUTHOR INFORMATION

Corresponding Author

*Phone: (602) 543-6087. Fax: (602) 543-6073. E-mail: Peter. [email protected]. Author Contributions ∥

REFERENCES

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M.C.H. and C.E.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the Thome Foundation and the U.S. National Institutes of Health for financial support of this work (R15 CA139364). We also thank Arizona State University’s Women and Philanthropy Grant Program for support. Thanks are also given to John Greaves of the High-Resolution Mass Spectrometry Laboratory at University of California, Irvine (UCI). Computer time was provided by USF Research Computing, sponsored in part by NSF MRI CHE-1531590 to A.v.d.V. Patent applications covering the technologies 8937

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